As methods of obtaining gene products of prokaryotic cells such as bacteria and blue-green algae, there have conventionally been used methods of culturing prokaryotic cells having said gene and then isolating and purifying the gene products of interest. However, when the gene product thus obtained is used as a vaccine, such methods had a problem of production efficiency in which an adequate amount of expression cannot be secured, and a safety problem due to difficulty in removing impurities such as pyrogens in the purification process.
Thus, focusing on the advantage of obviating the need of removing pyrogens, attempts have been made to introduce into a eukaryotic cell a recombinant vector in which the gene of interest derived from a prokaryotic cell has been integrated into a vector such as a virus, and then allowing the gene to be directly expressed in the eukaryotic cell. However, since prokaryotic cells and eukaryotic cells are essentially different in their mode of gene expression, gene products of a prokaryotic cell expressed in a eukaryotic cell could not exhibit the activity at a level equivalent to those produced in the prokaryotic cell, which sometimes resulted in an inadequate immunogenicity.
For example, U.S. Pat. No. 5,871,742 describes that an avipoxvirus vector which has integrated an antigen gene TTM-1 (TTM-1 gene) derived from Mycoplasma gallisepticum is effective as a vaccine to protect against Mycoplasma gallisepticum infection. Subsequently, it was found that the product (TTMG-1 antigen) of the TTM-1 gene is displayed on the cell membrane when the TTM-1 gene is expressed in the prokaryotic cell, whereas the TTM-1 product expressed in a eukaryotic cell is not displayed on the cell membrane of the eukaryotic cell and thereby is unlikely to exhibit the inherent immunogenicity. As a result of further study, in order to display the TTM-1 product on the surface of eukaryotic cells, a fusion gene was constructed in which a DNA encoding a virus-derived type II signal sequence such as signal sequence (hereinafter referred to as MDV gB signal) of gB of Marek's disease virus (MDV) has been ligated to said gene. By integrating this fusion gene into avipoxvirus and allowing it to be expressed, the TTMG-1 antigen was successfully displayed on the surface of the cell membrane, and thereby a vaccine that exhibits a higher activity of protecting against infection was obtained (International Patent Publication WO97/36924).
In most cases, proteins synthesized in the eukaryotic cell are different from those synthesized in the prokaryotic cell in that the former has sugar chains attached thereto.
Yoshida et al. (2000) constructed a recombinant avipoxvirus in which mgc3 (the mgc3 gene), a gene derived from Mycoplasma gallisepticum, other than the TTM-1 gene was integrated, and investigated the expression of the product (the MGC3 antigen) of the mgc 3 gene by immunoprecipitation. As a result, it was confirmed that N-linked sugar chains are not attached (N-glycosylated) to the MGC3 antigen having no MDVgB signal added thereto whereas the MDVgB signal-added MGC3 antigen undergoes N-glycosylation. Yoshida et al. (2000) also confirmed that the MGC3 fusion protein produced by a recombinant avipoxvirus that has integrated therein a fusion gene of the mgc3 gene and DNA encoding the MDVgB signal is 50-fold more reactive to a monoclonal antibody 35A6 that recognizes the MGC3 protein than the MGC3 antigen produced by a recombinant avipoxvirus that has integrated therein only the antigen gene mgc3. Based on this, it had been thought that though the fusion of a DNA encoding the MDVgB signal with the antigen gene may result in the addition of sugars to the protein obtained, a highly immunogenic protein could be obtained without N-glycosylation affecting immunogenicity.